N2A Titin: Signaling Hub and Mechanical Switch in Skeletal Muscle

Figure 1. (A) Layout of titin in skeletal muscle sarcomeres. Each titin molecule is bound to the thin filament (blue) in the Z-disk (blue triangles on left side of half-sarcomere) and to the thick filaments (purple) in the A-band. The N2A segment (red) is located between the proximal tandem Ig segments (orange) and the PEVK segment (green). Ig, immunoglobulin. Reproduced with permission from Nishikawa [21]. Copyright 2016, The Company of Biologists Ltd. (B) Layout of the N2A region. This region includes Ig80, unique N2A sequence (UN2A), Ig81, Ig82, and Ig83. The exons of the mouse Ttn gene are shown below, along with the location of the mdm deletion.

Figure 2. Schematic of the titin-N2A signaling complex. MARP1 and 2 bind competitively to N2A titin’s unique sequence (UNA), located between Ig80 and Ig81. CAPN3 binding is localized to both Ig80/UN2A and Ig82/Ig83. Myopalladin (MYPN) associates with the N-terminal domains of MARPs. Adapted from Miller et al. [41] and Hayashi et al. [43].

Figure 3. N2A—actin interaction eliminates low-force straightening of tandem Ig domains. (A) Passive sarcomere at slack length. (B) As the sarcomere is stretched passively beyond its slack length, the proximal tandem Ig segments unfold approximately to their contour length. (C) After the proximal tandem Ig segments have reached their contour length, further passive stretching extends the PEVK segment. (D) Upon activation, N2A titin binds to actin. (E) When the active sarcomere is stretched, only the PEVK segment (green) extends. Reproduced with permission from Nishikawa [21]. Copyright 2016, The Company of Biologists Ltd.

Figure 4. Activation increases titin-based force and stiffness of wild type psoas myofibrils, but not mdm myofibrils, during stretch. Stress (nN/μm2) vs. sarcomere length (μm) of single psoas myofibrils from the wild type (WT, black open symbols) and muscular dystrophy with myositis (mdm, gray filled symbols) mice during active (diamonds) and passive stretch (circles). There is no difference in passive stress between WT and mdm myofibrils. Actively stretched WT myofibrils are stiffer than active mdm and both passive mdm and WT, while actively stretched mdm myofibrils do not differ in stiffness from passively stretched mdm and WT myofibrils. Reproduced with permission from Powers et al. [25]. Copyright 2016, The Company of Biologists Ltd.

Figure 5. Residual force enhancement (A,B) and depression (C,D) increase with activation in wild type (blue) but not mdm (red) soleus muscles. The contribution of passive stress was subtracted from residual force enhancement and depression. Darker bars indicate maximal activation and lighter bars indicate submaximal activation. Reproduced with permission from Tahir et al. [80]. Copyright 2019, The Company of Biologists Ltd.

Figure 6. Length-dependence of Ca2+-sensitivity is similar in the wild type (black) and mdm (gray) fiber bundles. (A) Soleus and (B) extensor digitorum longus (EDL). Ca2+ sensitivity relationships at sarcomere lengths of 2.4 (dashed lines) and 3.0 µm (solid lines) from wild type and mdm fiber bundles (top). Individual data points for each curve are shown below. The shift in Ca2+-sensitivity from 2.4 to 3.0 µm was similar between genotypes. Reproduced with permission from Hessel et al. [26]. Copyright 2019, The Company of Biologists Ltd.